bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.432277; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 A drastic shift in the energetic landscape of toothed whale sperm cells 2 L. Q. Alves1†, R. Ruivo1†, R. Valente1,2, M. M. Fonseca1, A. M. Machado1,2, S. Plön3, 3 N. Monteiro2,4, D. García-Parraga5, S. Ruiz-Díaz6,7, M.L. Sánchez-Calabuig6,8, A. 4 Gutiérrez-Adán6*, L. Filipe C. Castro1,2*. 5 1CIMAR/CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, 6 University of Porto, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, 7 Portugal. 8 2FCUP - Department of Biology, Faculty of Sciences, University of Porto (U. Porto), 9 Rua do Campo Alegre, Porto, Portugal. 10 3Bayworld Centre for Research and Education (BCRE), Port Elizabeth, South Africa. 11 Present address: Hanse Wissenschaftskolleg, Institute for Advanced Study, 27753 12 Delmenhorst, Germany 13 4CIBIO - Research Centre in Biodiversity and Genetic Resources, Campus Agrário de 14 Vairão, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal 15 5Veterinary Services, L'Oceanográfic, Ciudad de las Artes y las Ciencias, Junta de Murs 16 i Vals, s/n, 46013 17 6Departamento de Reproducción Animal, INIA, Av. Puerta de Hierro, 18, 28040 18 Madrid, Spain. 19 7Mistral Fertility Clinics S.L., Clínica Tambre, 28002 Madrid, Spain 20 8Department of Animal Medicine and Surgery, Faculty of Veterinary Science, 21 University Complutense of Madrid, 28040, Madrid, Spain 22 *Correspondence to: [email protected], +351 223401800, ORCID ID: 0000- 23 0001-7697-386X; [email protected], +34 913473768, ORCID ID: 0000-0001-9893-9179 24 25 †Equal Contribution 26 27 L. Q. Alves: [email protected], +351 223401831, R. Ruivo: [email protected], 28 +351 223401818, R. Valente: [email protected], +351 223401800, M. M. 29 Fonseca: [email protected], +351 223401800, A. M. Machado: 30 [email protected], +351 223401831, S. Plön: [email protected], 31 +27-(0)76 3791067, +49-(0)4221 9160223, N. Monteiro: [email protected], 32 +351 252 660411, D. García-Parraga: [email protected], +34 960470647, S. 33 Ruiz-Díaz: [email protected], +34 913473768, M.L. Sánchez-Calabuig: 34 [email protected], + 34 913473765 35 36 37 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.432277; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 38 Abstract: Mammalia spermatozoa are a notable example of energetic 39 compartmentalization. While mitochondrial oxidative phosphorylation is restricted to the 40 midpiece, sperm-specific glycolysis operates in the flagellum. Consequently, these highly 41 specialized cells exhibit a clear adaptability to fuel substrates. This plasticity is essential 42 to ensure sperm motility, and is known to vary among species. Here we describe an 43 extreme example of spermatozoa-energetics adaptation. We show that toothed whales 44 exhibit impaired sperm glycolysis, due to gene and exon erosion, and demonstrate that 45 dolphin spermatozoa motility depends uniquely on endogenous fatty acid β-oxidation, 46 but not carbohydrates. Our findings substantiate the observation of large mitochondria in 47 spermatozoa, possibly boosting ATP production from endogenous fatty acids. This 48 unique energetic rewiring emphasizes the physiological body reorganisation imposed by 49 the carbohydrate-depleted marine environment. 50 Main Text: The rise of modern human societies from ancient civilizations implied a 51 division of labor and specialization: critical leverages towards the evolution of complex 52 interactions. Such trend is also noticeable in the evolution of biological complexity, 53 associated with the emergence of structured compartments and resource distribution 54 towards specific functions (e.g. multicellularity). At the cellular level, we can also 55 recognize structural partitions. Yet, the compartmentalization of metabolic pathways, in 56 time and space, within such structures is less intuitive (1). Mammalian spermatozoa 57 provide an illustrative example. The energy in the form of ATP production, vital for 58 motility, capacitation, and fertilization, is subcellularly separated in sperm cells. While 59 glycolysis provides a local, rapid, and low-yielding input of ATP along the flagellum 60 fibrous sheath, oxidative phosphorylation (OXPHOS), far more efficient over a longer 61 time frame, is concentrated in the midpiece mitochondria (2). The relative weight of 62 glycolysis and OXPHOS pathways in sperm function is species-dependent, and sensitive 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.432277; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 63 to oxygen and substrate availability (i.e., glucose, fructose, fatty acids, lactate, glycerol, 64 ketone bodies, or pyruvate) (3). For example, while OXPHOS was suggested to be 65 predominant in bull spermatozoa, in humans and mice, glycolysis is essential, notably in 66 the distal tail (4, 5). Besides partitioning energy production, sperm cell energetics display 67 an additional singularity: the occurrence of sperm-specific gene duplicates and alternative 68 spliced variants, although with conserved function (Fig. S1) (6). 69 The wider selective forces driving the compartmentalization and adaptability of this 70 energy system in mammalian species remain largely unknown; much like the impact of 71 ecosystem resource availability (e.g. carbohydrates, fatty acids, proteins) and dietary 72 adaptations in reproductive physiology traits (7). Here, we investigated the Cetacea, an 73 iconic group of fully aquatic and carnivorous marine mammals, evolutionarily related to 74 extant terrestrial herbivores. In this lineage, episodes of profound trait adaptation have 75 been accompanied by clear genomic signatures (8-10). To investigate the evolution of 76 sperm energetics, we started by analyzing the glycolysis rate-limiting enzyme, 77 glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (Gapdhs) (11), in 78 mammalian genomes. Using a pseudogene inference pipeline, Pseudochecker (12), we 79 scrutinized the coding condition of Gapdhs in 163 genomes and found that this gene 80 displays a Pseudoindex, a probability measure of pseudogenization (functional 81 inactivation) (12), compatible with gene lesion events in an extremely restricted number 82 of mammalian lineages (Table S1; table S2) (13). 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.432277; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 83 84 Fig. 1. Erosion of sperm-specific glycolysis genes. (A) The mutational landscape of 85 Gapdhs in Cetacea and hippopotamus (H. amphibius). Examples of the detected gene 86 disruptive mutations (insertions in purple, deletions in blue, in-frame premature stop 87 codons in red, and splice site mutations in yellow) are represented according to their 88 position within the affected exons (numerated on top, from the 3rd to the 10th exon – full 89 representation available in fig. S4). Odontoceti families are represented as follows: I. 90 Delphinidae, II. Phocoenidae, III. Monodontidae, IV. Lipotidae, V. Pontoporiidae, VI. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.01.432277; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 91 Iniidae, VII. Ziphiidae, VIII. Kogiidae, IX. Physeteridae. (B) Branch-specific relaxation 92 parameters inferred for Gapdhs in mammals. (C) Comparative synteny maps of the Pgk2 93 genomic locus in Cetacea and Bos taurus (cow). Orthologous genes are joined by lines 94 (fig. S28). (D) Aldoa_v2 exon erosion in Cetacea. C-Coding, N-Not found, Ψ- 95 Pseudogenized, Green-Expressed, Yellow-Not expressed. Silhouette images were 96 retrieved from http://phylopic.org/. 97 98 In effect, this pattern is almost exclusively limited to toothed whales (Odontoceti, 99 Cetacea). Next, we employed manual validation (Fig. S2) to inspect the coding sequence 100 of Gapdhs in species with a Pseudoindex higher than 2, including 18 species of toothed 101 whales (13), unveiling numerous gene-inactivating mutations in Odontoceti species (Fig. 102 1A; fig. S3-S21). Further examination of a bottlenose dolphin (Tursiops truncatus) sperm 103 transcriptome showed expression of Gapdhs with an aberrant splicing pattern, with 104 sequence reads encompassing the identified genome mutations (13) (fig. S22). A single 105 mutation is shared across all the examined Odontoceti species, with exception of the early 106 diverging beaked (Kogiidae)